Glycolipids are known to affect four general functions related to cell membranes; stabilization, shape determination, recognition and ion-binding (Curatoto, W: Biochim. Biophys. Acta, 906, 1987, 137-160). Glycolipids are also implicated for various immunological phenomena. The interference of gangliosides in the action, of interferon was reported in early seventies (Bensancon, F. and Ankel, I I Nature, 252, 1974, 478-480). Interleukin-2 was found to have affinity towards gangliosides (Parker, J., et at, FEBS Lett., 170, 1984, 391-395). It has been reported that the involvement of glycolipids in lymphocyte stimulation (Spiegel, S., et al., Proc. Natl. Acad. Sci., 76, 1979, 5277-5281; Spiegel, S and Wilchesk, M. J. Immunol, 127, 1981, 572-575; Spiegel, S. and Witcheck, M., Mol. Cell. Biochem. 55, 1983, 183-190). It was suggested that gangliosides might be involved in immuno regulations (Miller, H. C. and Essenman, W. J., J. Immunol. 115, 1975, 839-843); Chaney, W. G. et. al. Cell Immunol. 86, 1984, 165-170). The possibilities of immunosuppressive effects of gangliosides from tumor cells were also reported (Ladish, S, et. al., Cancer Research, 43, 1983, 3808-3813), Gangliosides are also known to have significant impact in the growth of neurons. A group of scientists reported that proliferation of neurites of felines with gangliosidosis was observed (Purpura, D. P. and Baker, H. J Brain Res., 143, 1978, 13-26). Later, these effects were elicited by various purified gangloslides (Byrene, M. C., et al, J. Neurochem., 41, 1983, 1214-1222). These in vitro results were utilized for curing neuropathies with ganglioside injections (Gorio, A. et al, in Ganglioside Structure, Function and Biomedical Potential, Ed. by Ledeen, R, Yu, R, Raport, M and Suzuki, K. Plenum Press, New York, 1984, 549-561). Clinical trials on human volunteers showed that ganglioside injections improve symptoms of diabetic neuropathy (Narden, A., et al., in Ganglioside Structure, Functions and Biomedical Potential, ed. By Ledeen, R, Yu, R., Rapoort, M and Suzuki, K, Plenum Press, New York, 1984, 593-600). In oncogenesis also, glycolipids play an significant role. It was observed that qualitative and quantitative changes in the cell glycolipids occur during differentiation and oncogenesis (Hakomori, S. Biochim. Biophysm Acta, 417, 1975, 55 89; Feizi, T.; Nature, 314, 1985, 53-57). The glycolipids from the tissues of the tumors found to have both qualitative and quantitative differences with normal tissues (Hamomori, S. and Murakami, W. T., Proc. Natl. Acad. Sci., USA, 59, 1968, 254-261). Different antibodies were used to characterize a number of tumor antigens on gangliosides (Tai, T., et al., Biochim. Biophys, Acta, 835, 1985, 577-583) and on neutral glycosphingolipids (Willson, K. R., et al. J. Biol. Chem, 1983, 4091-4097). These studies of glycolipid antigens on cell surfaces have generated significant information regarding glycolipid synthesis and the nature of the cell surface in differentiation and oncogenesis.
In recent years, many researchers have shifted their focus to understand the possible mechanisms of regulating various physiological phenomena by glycolipids. Biological activities of glycolipids isolated from various natural resources are being evaluated. Scientists are also checking the suitability of use of glycolipids in pharmaceutical and cosmetic formulations. The use of glycolipids from sivers sagebrush (wormwood) as organoleptic and biologically active additive in food industries were tested (Gubanenko, G. A., et al, Pischch. Prom. St. 6, 1998, 26-27). The possible role of glycolipids in type 1 diabetes and lupus, in intracellular bacterial infections and in tumor rejections were also investigated (Park, S. II. et al, Semin. Immunol., 10(5), 1998, 391-398). Biological activities of some natural and synthetic glycolipid antigens that induce a CD-1 restricted 1-cell response are described in PCT Int, Appl. WO 99, 12562 (Porcelli, S. A. and Moody, D. B., 1999). Glycolipids of these kinds were used for treatment of people infected with Mycobacterium leparae. Glycolipids also have an important role in lens fiber developed in human eye (Ogiso, M, Acta, Biochim. Pol., 45 (20, 1998, 501-507).
The transfection efficiency and cell uptake of DNA/glycolipid complexes were considered as potential HIV-1 fusion cofactor (Djilalj, H., et al, Biochim, Biophys. Res. Commun. 246(1), 1998, 117-122). Glycolipid-based compositions were used for controlling of colonization of bacterial plaque in the oral cavity (Bundy, J. A. U.S. Pat. No. 5,871,714, 1999). Another formulation consisting of glycolipids was used in treatment for prophylaxis or acidic gut syndrome (Rowe, J. B., PCT Int. Appl. WO 99, 00136, 1999). The role of glycolipids on age related changes of the brain as well as in Alzheimer's disease was examined in a recent review (Endo, T. Tampakskitu Kakusan Koso, 43 (16), 1998, 2582-2588). Sophorolipids were isolated from various sources (Marchal, R., et al., U.S. Pat. No. 5,900,366, 1999: Daniel; H. S. et al, Biotechnol., 51 (1), 1999, 40-45) including from deproteinized whey and rapeseed oil (Daniel, H. S., et al, Biotechnol, Lett, 29(12), 1998, 1153-1156). Bioactivity of these sophorolipids and sophorolipid-derivatives were evaluated (Sholtz., C., et al., Polym, Prepr. 39 (2), 1998, 168-169). It was observed that glycolipids like sophorolipids exhibit cell growth inhibition properties for. Jurkat (Leukemia) and Tu-138 (head and neck cancer) cells. Glycosylphosphatidyl inositols have an effect on material toxins and in release of cytokines like tumor necrosis factor- and interleukin-10 (Schmidt, A., et at, Exp. Parasitol., 88(2)., 1998, 95-102).
In the late nineties, a group of scientists isolated a new glycolipid from Gigartina tenella (Ohra K, et al., Jpn. Kokai Tokkyo Koho, JP 11, 106, 395, 1999). This glycolipid was found to have high activity as DNA-synthetase B-inhibitor, HIV inhibitor and as immunosuppressant. It was also found that these glycolipids can act as anti-cancer agents and have other immunosuppressive effects (Yoshida, M., et al., Jpn Kokai Tokkyo Koho J P, 10, 152, 498, 1998). The cellular responses of Clusters of glycolipids and glycosyl-phosphatidyl inositol-anchor proteins in lymphoid cells towards raft patch formation in the Jurkat (Leukemia) T-cell lines were investigated (Harder, T. and Simons, K., Eur., J. Immunol, 29 (2), 1999, 556-562). Growth factor-induced release of a glycosyl-phosphatidyl inositol (GPI)-linked protein from HEP-G2 human carcinoma cell lines was examined earlier (Roberts, J. M., et, al., FEBS Lett., 267 (2), 267 (2), 1996, 213-216). The possible use of glycolipids in gene therapy was also tested (Havermann, K., et. al, EP 893,493, 1999).
It is surprising that these compounds are not exploited commercially though they have such diversified functions and biological activities. The most probable reasons could be the lack of availability of natural sources rich in glycolipids or may be non availability of commercially viable processes for their isolation. Rice bran oil may offer significant possibilities as its glycolipids content was found to be quite high. Earlier, a process was developed in this laboratory to isolate and purify glycolipids from rice bran oil [Vali et. al. U.S. Pat. No. 6,953,849 (2005)].
In this present invention, glycolipids and phospholipids isolated from rice bran oil gums were used either separately or in combination to prepare a non-viral formulation for a potent carrier to deliver anti-cancer genes Et bioactive compounds to breast and lung cancer cells. There is an example of glycolipids (mannosylerythritol) originally isolated from Candida antarctica that was being supplemented in a cationic lipid formulation for the purpose of gene delivery to mammalian cells. (Kitamoto et. al. J. Bioscience and Bioengineering, 2002, 94, 187-201). The major components present in rice bran glycolipids are acylated steryl glucoside (ASG), digalactosyldiatyl glycerol (DGDG) and monogatactosylnionoacylglycerot (MGMG). The major components of rice bran phospholipids are phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidic acid (PA) (Hemavathy J and Prabhakar J V, J. Amer. Oil Chem. Soc., 1987, 64, 1016-1019).
In the present invention, the glycolipid and phospholipids are either individually associated with respective cationic lipid formulation or they are supplemented to the cationic liposomal formulation in the form of cocktail mixture. This not only increased the transfection efficacy of cationic lipid but also the new formulation mediated gene transfer to cancer cells only. The same glycolipid/phospholipid associated or supplemented cationic liposomal formulation did not show efficient transfection in cells with no cancer lineage. This potent liposomal formulation can be used for specific delivery of anticancer therapeutics such as anticancer genes to cancer cells only without eliciting any treatment related toxicity to normal cells.
Chemotherapy and radiation therapies are two current clinical modalities commonly used for the treatment of cancer. Mostly these techniques are effective to block the growth of tumor; however, there is often a recurrence of the disease, possibly because of incomplete cell killing or cells acquiring drug resistance. Moreover, none of the current treatment modalities are hundred non-toxic to normal cells and are often deleterious to general health.
In comparison to the non-viral gene delivery method as depicted in the present invention, the viral based gene delivery is also quite well known and is extensively investigated utilizing their phenomenally efficient process of delivering genes to wide variety of cells. A number of problems including host toxicity, immunogenic responses and non-specific genomic integration of transferred gene make viral delivery a risky option for delivering genes. In comparison, non-viral gene delivery is a much more robust and clinically safe option compared to viral counterparts. The patented cationic lipid, DODEAC [Banerjee et. al. U.S. Pat. No. 6,333,433 B1 (2001), U.S. Pat. No. 6,346,516 B1 (2002), U.S. Pat. No. 6,503,945 (2003a), U.S. Pat. No. 6,541,649 (2003b)], whose structure is. N,N-dihydroxyethyl, N,N-dioctadecyl, ammonium chloride forms cationic liposome using co-lipid cholesterol in membrane filtered water. This product has been, used for the transfection of DNA into cultured eukaryotic cells of various origins. However, the formulation, in spite of exhibiting moderate transfection of genes to all cells irrespective of origin, shows no specific targeting of genes to cancer cells. Towards this end, the present invention relates to development of a new rice-bran isolated glycolipid/phospholipid carrying cationic lipid based formulation, which targets and deliver genes to breast and lung cancer cells alone. The idea of making this formulation stems from the fact that glycolipids, which has close structural resemblance with galactosyl- or other carbohydrate ligands that show highly selective affinity to asialoglycoprotein or other carbohydrate receptors that are avidly expressed in various cancer cells.